The present invention relates, as indicated, to a competitive radioisotopic enzyme or biochemical cofactor assay for measuring the concentration of folate in serum. The term "folate" is well understood by those skilled in biochemistry. It is generic and includes not only the derivatives of pteroylmonoglutamic acid but also the salts and other neutralization products thereof. Whether a material is in the acidic or basic form will depend upon the pH of the medium in which it is found. Thus, the term folate includes substituted derivatives of pteroylmonoglutamic acid and the polyglutamate forms as well as esters, salts and other such derivatives involving not only the carboxyl group but also other points of substitution on the molecule. It is often helpful to know the serum folate concentration in order to confirm the etiology of a patient's anemia. Presently known microbiological assays for measuring the serum folate concentration, however, are time-consuming and, like other microbiological assays, are affected by antimicrobial and antineoplastic agents which may be present in the serum sample. Reference may be had to the work by Herbert entitled, "Asceptic Addition Method for Lactobacillus Casei of Folate Activity in Human Serum," J. Clin. Path. 19: pp 12-16, 1966; and Grossowicz et al, "Microbiologic Determination of Folic Acid Derivatives in Blood," appearing in Blood 20, pp 609-616, 1962. The present process is distinguished from these microbiological assays in utlizing a radio assay procedure characterized by a direct competitive binding between a labeled folate and a second folate, in one case the second folate being known and in the second case the second folate being a serum folate, whereby the concentration of folate in the serum may be determined. This assay may be completed in a morning's work and the results obtained by the end of the day. This assay employs .sup.3 H-pteroylmonoglutamate (hereinafter identified as .sup.3 H-PGA) as the labeled folate, d,1,5-methyltetrahydrofolic acid (hereinafter called 5CH.sub.3 FH.sub.4) as a known folate, a partially purified folate binder extracted from hog kidney or other suitable animal organ. This assay is sensitive to 25 picograms of 5CH.sub.3 FH.sub.4, the main serum folate (see Herbert et al, "Studies on the Identification of a Folate Compound of Human Serum," J. Clin. Invest. 41: pp 1134-1138, 1962). Unlike microbiological assays, the present assay is unaffected by antimicrobials or antineoplastic agents such as Methotrexate (amethopterin), 4-(amino-N.sup.10 -methylpteroylglutamic acid). Reference may also be had to Rothenberg et al, "A Radio Assay for Serum Folate: Use of a Two-Phase Sequential-Incubation, Ligand-Binding System," New England Journal of Medicine 286: 1335-1339, 1972.
Binders of pteroylmonoglutamate (PGA) and some of its derivatives have been described in milk, serum from some pregnant women, folate deficient patients, and serum from some patients with a malignant disease. Folate binders may be extracted from various animal organs, particularly the kidneys and pancreas. Spleen, liver, heart and muscle tissue, while they do contain minor amounts of material useful as binder, do not contain such material in commercially significant amount. The procedure of extraction is in any case the same. Best results have been secured with hog kidney binder factor. Accordingly, the description of the procedure for extracting the binder factor from animal organs will be exemplified by reference to the procedure for extracting binder factor from hog kidneys. It will be understood that the same procedure may be utilized for preparation of binder factors from such other organs.
The hog kidney binder preparation has proved very useful in setting up a competitive ligand binding assay for measuring folate, especially serum folate. In addition, this binder appears to offer a good model system for study and comparison to folate binders found to be in the serum of patients with certain diseases.